Coupled transcription and translation in mammalian and avian cell-free systems

Abstract

In cell-free extracts of mouse L cells and of primary chick embryo cells, purified vesicular stomatitis virus (VSV) directed coupled transcription and translation of the viral genes. The proportions of virus and cell extract were equivalent to a multiplicity of infection of about 20 PFU per cell. The reactions, which continued for at least 8 hr, resulted in the synthesis of polypeptides which resembled the authentic viral polypeptides N, NS, and M by the criteria of their sizes and tryptic peptide fingerprints. In addition, a minor protein product of the cell-free systems yielded tryptic peptides characteristic of the viral G protein, although its apparent size was slightly less than that of authentic G protein. No product corresponding to the viral L protein was detected. The RNA products of the coupled system were characterized by affinity chromatography on oligo(dT)-cellulose and by polyacrylamide-gel electrophoresis under denaturing conditions. Four size classes of poly(A)-containing RNA were detected, with apparent molecular weights of 2.15, 0.74, 0.55, and 0.3 × 10 6. These contained the messenger RNAs for the viral L, G, N, and NS + M proteins, respectively. At later times during transcription in vitro, synthesis occurred of increasing amounts of four species of RNA which lacked poly(A), and which had correspondingly lower apparent molecular weights. These species were synthesized de novo, rather than by cleavage of the poly (A) tracts from the RNA made at early times. No RNA corresponding in size to the viral genome was synthesized. The remarkable longevity of the cell-free protein synthesis prompted a study of the structural and functional stability of the VSV messenger RNAs synthesized in the L-cell system. Chase experiments showed that the poly(A)-containing and poly(A)-lacking RNA species were degraded at approximately the same rate, with half-lives of about 12 hr. The methyl groups in the 5′-terminal cap structures of the poly(A)-containing messenger RNAs had a similar half-life. Moreover, preformed VSV messenger RNA added exogenously to the cell-free system at the same concentration as in the coupled system showed a similar rate of degradation. The functional stability of the messenger RNA was investigated by assaying protein synthesis after the addition of S-adenosylhomocysteine to the coupled system to inhibit the synthesis of further translatable messenger RNA. The results showed that the pronounced stability of messenger RNA at low concentration, rather than its continued synthesis, accounted for the longevity of protein synthesis in this system.